Integrative single-cell RNA-seq and TCR repertoire analysis reveals distinct immunological features of acute liver allograft rejection

Liver transplantation is the definitive treatment for end-stage liver disease, but acute allograft rejection affects 15–30% of recipients. Early prediction of rejection remains difficult because the underlying subclinical immunological mechanisms are incompletely understood, and diagnosis still relies on invasive histological biopsy. This study aimed to identify molecular signatures predisposing recipients to acute rejection. We analyzed paired pre- and post-transplant peripheral blood mononuclear cell samples from 20 liver transplant recipients, including 10 with acute rejection (AR) and 10 without rejection (NR). Cases were matched for clinical parameters, including age, sex, HCC status, autoimmune status, ABO compatibility, and donor age. Using integrated single-cell RNA and T cell receptor sequencing, we profiled 88,940 high-quality cells and characterized cellular heterogeneity, clonal dynamics, gene regulatory networks, and intercellular communication to identify rejection-associated immune signatures. AR recipients exhibited distinct pre-transplant immune states, indicating molecular features predisposing to acute rejection. These included inflammatory NK cell activation with enhanced interferon responsiveness, altered monocyte composition and transcriptional programs marked by interferon-associated signatures, and skewing toward effector memory CD8 T cell states relative to NR recipients. Following transplantation, AR patients maintained heightened immune activity despite immunosuppression, accompanied by expansion of KIR-expressing effector memory CD8 T cell populations and increased IRF1 expression in CD8 T cells. We further identified 15 robust AR-associated genes consistently detected across time points and validated a subset of these genes in liver biopsy tissue. Recipients who developed acute rejection exhibited distinct pre-transplant immune states and underwent differential immune remodeling after transplantation compared with non-rejecting recipients. These rejection-associated cellular and molecular signatures may enable early, non-invasive identification of patients at risk and provide a basis for more precise monitoring and management in liver transplantation.